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Original Article

Validation of an HPLC-MS method for the determinatin of vardenafil in rat urine

Liudmila Osypchuk, Iryna Halkevych, Sophia Davydovych, Yuriy Bidnychenko.

Cited by 5 Articles

Summary: A simple and accurate technique of high-performance liquid chromatography with mass spectrometry was developed for the quantitative determination of vardenafil and its metabolites in urine. Extraction of vardenafil and internal standard (sildenafil) was performed from 5 ml of urine with 1,2-dichloroethane at pH=7.5, followed by purification of samples on Oasis HLB cartridges. Chromatographic separation was carried out on a Zorbax SB-C18 reversed-phase column (50 mm x 2.1 mm) in linear gradient mode, at a rate of 0.4 ml/min. Mobile phase composition: 0.1% aqueous formic acid solution and 0.1% formic acid solution in acetonitrile. Detection of vardenafil and sildenafil in samples was performed using a single quadrupole mass spectrometer with electrospray ionization. Mass spectral analysis was performed in the m/z range: 50-500 with the fragmentation of 50 m/z under positive ionization. The linear dependence for vardenafil was in the range of 7-500 ng/ml; LOD and LOQ were 5 and 7 ng/ml, respectively. The developed method was tested on specimens of rat urine taking vardenafil at a dose of 0.28 mg/kg. 11.27-13.60 ng of vardenafil were detected in 1 ml of daily urine of the animals. This method is recommended for use in toxicological practice.

Key words: vardenafil, HPLC, mass spectrometry, urine.

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